title: Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia.
abstract: The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.
authors.affiliation: Inserm U954 Nutrition-Genetics-Environmental Risk Exposure and Reference Centre of Inborn Metabolism Diseases, Medical Faculty of Nancy University and University Hospital Centre, Nancy, France.
authors.forename: Ma'atem B
authors.initials: MB
authors.lastname: Fofou-Caillierez
authors.forename: Nadir T
authors.initials: NT
authors.lastname: Mrabet
authors.forename: Céline
authors.initials: C
authors.lastname: Chéry
authors.forename: Natacha
authors.initials: N
authors.lastname: Dreumont
authors.forename: Justine
authors.initials: J
authors.lastname: Flayac
authors.forename: Mihaela
authors.initials: M
authors.lastname: Pupavac
authors.forename: Justine
authors.initials: J
authors.lastname: Paoli
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Alberto
authors.forename: David
authors.initials: D
authors.lastname: Coelho
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Camadro
authors.forename: François
authors.initials: F
authors.lastname: Feillet
authors.forename: David
authors.initials: D
authors.lastname: Watkins
authors.forename: Brian
authors.initials: B
authors.lastname: Fowler
authors.forename: David S
authors.initials: DS
authors.lastname: Rosenblatt
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Guéant
mesh_terms: D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000749:Anemia, Megaloblastic / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D006879:Hydroxocobalamin / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010088:Oxidoreductases; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism; D014806:Vitamin B 12 Deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D020033:Protein Isoforms; C019476:mecobalamin; C508359:MMACHC protein, human; D010088:Oxidoreductases; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12; D006879:Hydroxocobalamin